1. Field of the Invention
The present invention relates generally to scanning confocal microscopes and, more particularly, to microscopes of this kind that process data acquired by scanning a sample with ultraviolet light and detecting the resulting fluorescence and that provide a continuous display of scan data.
2. Description of Related Art
In the scientific fields of physiology, cytobiology, etc., it is important to investigate the behavior of the intracellular ions of calcium, sodium, magnesium, etc. This is because these ions are thought to be closely linked with intracellular physioactivation. As part of a method of research into the behavior of the intracellular ions, fluorophores commonly are injected into cells. Such fluorophores combine uniquely with the certain species of ions within the cells and fluoresce when irradiated with excitation light of specified wavelength, visible or ultraviolet.
By way of example, fluorescent probes indo-1, fura-2, fluo-3 and rhod-2 are known fluorophores useful in the detection of the calcium ions. Any of these fluorescent probes can be used to detect the presence of calcium ions within the cells. For example, the probe indo-1 fluoresces at wavelengths of either 405 nanometers (nm) or 485 nm in accordance with the concentration of the calcium ions, in response to excitation by ultraviolet radiation having a wavelength of about 350 nm. The probe fura-2, on the other hand, fluoresces at a wavelength of about 500 nm, in response to excitation by ultraviolet radiation having a wavelength of about 340 nm or 380 nm.
When the fluorescent probe has combined with the calcium ions and is excited by the ultraviolet light, it fluoresces in an amount that varies in accordance with the calcium ion concentration. Therefore, the concentration of the calcium ions in each local area of a sample can be determined by measuring the intensity of the fluorescent light. The excitation and fluorescence detection can be carried out across sample surface, whereby a two-dimensional video image can be obtained. Further, a plurality of video images can be obtained in time series, whereby the time behavior of the ions can be investigated in detail.
Where the ratio between the intensities of the two peaks of the fluorescence spectra is detected under the ultraviolet excitation of the fluorescence probe indo-1, or where the ratio between the intensities of the respective peaks of the fluorescence spectra is detected under the alternate excitation operations of the fluorescent probe fura-2 with the two wavelengths of ultraviolet radiation, an accurate measurement of calcium ion concentration can be reliably obtained.
One suitable confocal microscope for scanning a sample detecting the resulting fluorescence is disclosed in copending and commonly-assigned U.S. patent application Ser. No. 07/862,633, filed Apr. 1, 1992 and entitled "Scanning Confocal Microscope." The disclosed microscope produces sets of digital data words that represent a succession of two-dimensional images of the sample in the two fluorescent wavelengths.
The scanning confocal microscope disclosed in the copending application is highly effective in generating data representing images of a sample's fluorescence. However, there still is a need for a scanning confocal microscope of this kind that further includes a processor for appropriately processing and transforming the data into a format that optimally conveys pertinent dynamic information about the sample and allows the user to selectively manipulate the data to accommodate the need for specific information. The present invention fulfills this need.